Mechanisms underlying the complex interrelationships of infection and graft survival during induction and maintenance of transplantation tolerance are not well understood. Our goal is to understand how infection blocks the induction of peripheral and central tolerance. We have developed several innovative technologies for identifying virus-immune T cells and determining their anti-viral and cross-reactive alloreactivity at the single cell level. We have also developed methods for 1) quantifying alloreactive T cells using a 'synchimera' model based on CD8+ TCR Tg mice, 2) identifying naive and effector alloreactive T cells by their rapid production of cytokines following alloantigen stimulation, and 3) quantifying in vivo CDS T cell effector function using an in vivo cytotoxicity assay. We will use these techniques with an exciting new technology for in vivo delivery of siRNA to block of CD40-CD154 interaction. These new technologies will allow us to test our overall hypothesis that induction of pro-inflammatory cytokines and IFN1 is a fundamental mechanism by which innate immune activation modulates the induction of peripheral and central tolerance. Specific Aim 1 is to determine mechanisms by which TLR ligation or virus infection modulates the induction of peripheral tolerance. We will test the hypothesis that innate immune activation by TLR agonists or virus infection abrogates the induction of peripheral tolerance through the production of pro-inflammatory cytokines and IFN1. Specific Aim 2 is to determine mechanisms by which TLR ligation or virus infection modulates establishment of hematopoietic chimerism and central tolerance. We will test the hypothesis that the induction of peripheral and central tolerance involves multiple different but overlapping mechanisms. This project should reveal the mechanism(s) by which infection compromises the induction of peripheral and central transplantation tolerance. This Project will interact closely with Project 2 studying the maintenance of tolerance, and Project 3 studying how alloreactive CD8 T cells die by apoptosis following costimulation blockade. These discoveries will be translated to human immune systems in Project 4 using both the Viral and Technology Core and Animal Core as critical resources for the accomplishment of our research goals.